This project proposes a combined theoretical and experimental approach to study the mechanism of enzymes that change DNA topology: type II DNA topoisomerases and site specific recombinases. DNA topoisomerases are targets of many antibacterial and anticancer drugs and therefore the knowledge of the mechanism of their action is very important for development of a new, potentially more specific therapies. The recombinases play an important role in the development of many virus diseases and some of them are important for knock-out technologies. A comprehensive understanding of the action of these enzymes requires an analysis of the DNA-protein systems beyond the scale of enzyme-DNA complexes, since the reaction outcomes also depend on global DNA conformations. The computer simulation of large-scale DNA conformational properties that we have developed in the previous funding periods allows us to predict the probabilities of different reaction outcomes for any hypothetical model of enzyme action and to compare the results with the corresponding measured values. This comparison serves as a stringent test of a given model. Specifically, we will focus on understanding (I) how type II DNA topoisomerases simplify of DNA topology, (ll) how DNAgyrase introduces exclusively negative supercoils in circular DNA, and (III) what determines the topological complexity of circular DNA molecules formed by different members of the integrase family of site-specific recombinases. The project is based on tight complementation between the experimental and theoretical methods. We found using our computer simulations that bending of the double helix upon enzyme binding should strongly affect the product distributions created by topoisomerases and recombinases. To address this issue experimentally we will develop a new technique to measure DNA bending in the complexes. This method will be applied to complexes of DNA with type II topoisomerases and site-specific recombinases. It will be also used to determine the sequence dependence of DNA bending rigidity. The results of this research will extend our understanding and DNA large-scale conformational properties, as well as to clarify mechanisms of some crucially important enzymes. New computational methods will be developed in the project which will allow us to estimate probabilities of very rare events, related to juxtaposition of specific DNA sites. [unreadable] [unreadable]